Techniques of making a predetermined change at a predetermined location in a target nucleic acid sequence of a cell have been described. These techniques utilize the cell's enzymes that concern DNA repair and homologous recombination. In these techniques an oligonucleotide or oligonucleotide analog is synthesized that contains two regions that have the sequence of the target gene that flank a region, termed a "mutator region," that differs from the target gene. In this application such oligonucleotides and analogs will be generically termed "mutational vectors." Such mutational vectors can introduce predetermined genetic changes into a target gene by a mechanism that is believed to involve homologous recombination and/or nucleotide excision and repair.
U.S. Pat. Nos. 5,565,350 and 5,731,181 to Kmiec describe mutational vectors that contain complementary strands wherein a first strand comprises ribonucleotide analogs that form Watson-Crick base pairs with deoxyribonucleotides of a second strand. Commonly assigned U.S. patent application Ser. No. 09/078,063, now U.S. Pat. No. 6,004,804, filed May 12, 1998, describes certain improvements in duplex mutational vectors, including a variant in which the mutator region is present on only one of the two strands. The use of Kmiec type mutational vectors in mammalian systems is described in U.S. Pat. No. 5,760,012 and in conjunction with macromolecular carriers in patent publication WO 98/49350 to Kren et al., and in related U.S. patent application Ser. No. 108,006.
Additional descriptions of the use of Kmiec type mutational vectors can be found in scientific publications Cole-Strauss et al., 1996, Science 273, 1386, Scientific publications concerning Kmiec type mutational vectors and macromolecular carriers include Kren et al., 1998, Nature Med. 4,285; Bandyopadhyay et al., April 1999, J. Biol. Chem. 274,10163.
The use of Kmiec type mutation vectors in plant cells is described in patent publications WO 99/25853 to Pioneer Hi-Bred International. WO 99/07865 to Kimeragen and WO 98/54330 to Zeneca Ltd. Scientific publications that describe the use of Kmiec type vectors in plants are Beetham et al., July 1999 PNAS 96, 8774 and Zhu, et al., July 1999, PNAS 96,8768.
The use of Kmiec type mutational vectors and variants thereof, which are double stranded is described in U.S. patent application Ser. No. 09/078,063, now U.S. Pat. No. 6,004,804, filed May 12,1998 to R. Kumar and R. Metz. The application of Kumar and Metz inter alia teaches that Kmiec type vectors and the variants thereof can be used in bacterial cells.
The use of single stranded oligodeoxynucleotides as mutational vectors to effect changes in a chromosomal gene in the yeast, S. cerevisiae, was described in reports from the laboratory of Dr. F. Sherman, Yale University. Moerschell, R. P., et al., 1988, Proc. Natl. Acad. Sci., 85, 524-528 and Yamamoto, T., et al., 1992, Yeast 8, 935-948 The optimum length of the mutational vectors used in these studies was 50 nucleotides.
An isolated report of the use of a 160 NT single and double stranded polynucleotide to attempt to make alterations in a chromosomal gene can be found at Hunger-Bertling, 1990, Mol. Cell. Bioch.92, 107-116. The results for single stranded polynucleotides were ambiguous because only the product of the experiments using double-stranded polynucleotides were analyzed.
The use of single stranded DNA fragment of 488 bp to make specific genetic changes in the cystic fibrosis transmembrane conductance regulator gene has been reported by Gruenet and colleagues. Goncz et al., November 1998, Hum. Mol. Genetics 7, 1913; Kunzelmann et al., 1996, Gene Ther. 3, 859-67.
Single stranded oligodeoxynucleotides of about 40 nucleotides in length in mammalian cells were used as a control for studies of episomal genes in which the oligodeoxynucleotide was covalently linked to a triplex forming oligonucleotide and that the oligodeoxynucleotide alone resulted in rates of predetermined genetic change of the episomal gene of about 1 per 5.times.10.sup.4, or fewer. Chan et al., April 1999, J. Biol. Chem. 74, 11541-11548, An earlier report of the use of single-stranded oligodeoxynucleotide to make predetermined changes in an episomal gene in a mammalian cell can be found in Campbell, C. R., et al., 1989, The New Biologist 1, 223-227
One aspect of the invention concerns oligodeoxynucleotides that have been modified by the attachment of an Indocarbocyanine dye. Indocarbocyanine dyes are known as excellent fluorophores. The synthesis of blocked indocarbocyanine .beta. cyanoethyl N,N-diisopropyl phosphoroamidites that are suitable for use in solid phase nucleotide synthesis is described in U.S. Pat. Nos. 5,556,959 and 5,808,044.
A second aspect of the invention concerns a composition comprising a single stranded oligonucleotide encoding a predetermined genetic change and a macromolecular carrier that comprises a ligand for a receptor on the surface of the target cell. A composition comprising a poly-L-lysine, a ligand for the asialoglycoprotein receptor and an antisense oligodeoxynucleotide of between 21 and 24 nucleotides is described in patent publication WO 93/04701 to Wu, G. Y.
A third aspect of the invention concerns a modification of a oligodeoxynucleotide by the attachment of a 3'-3' linked nucleotide. U.S. Pat. No. 5,750,669, assigned to Hoechst A. G., teaches such a modified oligodeoxynucleotide.